Phase-separated SOX5 drives human cranial neural crest fate commitment

Defective development of multipotent cranial neural crest cells (CNCCs) underlies diverse human neurocristopathies; yet, the mechanisms governing human CNCC fate commitment remain elusive. Using a human pluripotent stem cell (hPSC)-derived CNCC model, we generate transcriptomic, chromatin-accessibility, and transcription-factor-wide (TFome) CRISPR screening datasets, identifying SOX5 as a molecular switch for human CNCC specification. SOX5 loss retains the cells in a precursor-like state and strongly compromises their progression toward migratory CNCCs. Mechanistically, SOX5 forms nuclear condensates with properties consistent with liquid-liquid phase separation (LLPS) and compartmentalizes active chromatin and their three-dimensional organization. Through a prion-like domain (PLD), SOX5 self-associates and assembles a multiprotein hub containing the chromatin factor KU80, promoting a pro-CNCC program including ZNF467, EGR1, and SOX11. Disrupting the PLD shifts condensates to a more liquid-like state and disrupts KU80 recruitment. These findings uncover an LLPS-associated mechanism in human CNCC fate control, shedding light into the molecular etiology of neurodevelopmental disorders.